Dog Retina Research Articles

Structural Organization and Expression Pattern of the CanineRPGRIP1Isoforms in Retinal Tissue

To examine the structure and expression of RPGRIP1 in dog retina. Determination of the structural analysis and expression pattern of canine RPGRIP1 (cRPGRIP1) was based on cDNA amplification. Absolute quantification of the expression level of cRPGRIP1 splice variants was determined by qRT-PCR. Regulatory structures were examined by computational analysis of comparative genomics. cRPGRIP1 encompasses 25 exons that harbor a 3627-bp open reading frame (ORF) encoding a 1209-amino-acid (aa)-predicted protein. In addition to the main transcript, five full-length and several partial cRPGRIP1 isoforms were identified revealing four alternative 3'-terminal exons--24, 19a, 19c, and 19d--three of which could potentially produce C-terminally truncated proteins that lack the RPGR-interacting domain. A complex organization of the 5'-UTR for the cRPGRIP1 splice products have been described, with a common promoter driving multiple isoforms, including four full-length transcripts using the 3'-terminal exon 24. In addition, a potential alternative internal promoter was revealed to initiate at least two cRPGRIP1 splice variants sharing the same 3'-terminal exon 19c. Transcription initiation sites were highly supported by conserved arrangements of cis-elements predicted in a bioinformatic analysis of orthologous RPGRIP1 promoter regions. The use of alternative transcription start and termination sites results in substantial heterogeneity of cRPGRIP1 transcripts, many of which are likely to have tissue-specific expression. The identified exon-intron structure of cRPGRIP1 isoforms provides a basis for evaluating the gene defects underlying inherited retinal disorders in dogs.

Heterogeneity of Glia in the Retina and Optic Nerve of Birds and Mammals

We have recently described a novel type of glial cell that is scattered across the inner layers of the avian retina [1]. These cells are stimulated by insulin-like growth factor 1 (IGF1) to proliferate, migrate distally into the retina, and up-regulate the nestin-related intermediate filament transitin. These changes in glial activity correspond with increased susceptibility of neurons to excitotoxic damage. This novel cell-type has been termed the Non-astrocytic Inner Retinal Glia-like (NIRG) cells. The purpose of the study was to investigate whether the retinas of non-avian species contain cells that resemble NIRG cells. We assayed for NIRG cells by probing for the expression of Sox2, Sox9, Nkx2.2, vimentin and nestin. NIRG cells were distinguished from astrocytes by a lack of expression for Glial Fibrilliary Acidic Protein (GFAP). We examined the retinas of adult mice, guinea pigs, dogs and monkeys (Macaca fasicularis). In the mouse retina and optic nerve head, we identified numerous astrocytes that expressed GFAP, S100β, Sox2 and Sox9; however, we found no evidence for NIRG-like cells that were positive for Nkx2.2, nestin, and negative for GFAP. In the guinea pig retina, we did not find astrocytes or NIRG cells in the retina, whereas we identified astrocytes in the optic nerve. In the eyes of dogs and monkeys, we found astrocytes and NIRG-like cells scattered across inner layers of the retina and within the optic nerve. We conclude that NIRG-like cells are present in the retinas of canines and non-human primates, whereas the retinas of mice and guinea pigs do not contain NIRG cells.

Comparative study of Pax2 expression in glial cells in the retina and optic nerve of birds and mammals

Little is known about the expression of Pax2 in mature retina or optic nerve. Here we probed for the expression of Pax2 in late stages of embryonic development and in mature chick retina. We find two distinct Pax2 isoforms expressed by cells within the retina and optic nerve. Surprisingly, Müller glia in central regions of the retina express Pax2, and levels of expression are decreased with increasing distance from the nerve head. In Müller glia, the expression levels of Pax2 are increased by acute retinal damage or treatment with growth factors. At the optic nerve, Pax2 is expressed by peripapillary glia, at the junction of the neural retina and optic nerve head and by glia within the optic nerve. In addition, we assayed for Pax2 expression in glial cells in mammalian retinas. In mammalian retinas, unlike the case in chick retina, the Müller glia do not express Pax2. Pax2-expressing cells are found in the optic nerve and astrocytes within the mouse retina. By comparison, Pax2-positive cells are not found within the guinea pig retina; Pax2-expressing glia are confined to the optic nerve. In dog and monkey (Macaca fascicularis), Pax2 is expressed by astrocytes that are scattered across inner retinal layers and by numerous glia within the optic nerve. Interestingly, Pax2-positive glial cells are found at the peripheral edge of the dog retina, but only in older animals. We conclude that the expression of Pax2 in the vertebrate eye is restricted to retinal astrocytes, peripapillary glia, and glia within the optic nerve.

Lipid second messengers and related enzymes in vertebrate rod outer segments

Rod outer segments (ROSs) are specialized light-sensitive organelles in vertebrate photoreceptor cells. Lipids in ROS are of considerable importance, not only in providing an adequate environment for efficient phototransduction, but also in originating the second messengers involved in signal transduction. ROSs have the ability to adapt the sensitivity and speed of their responses to ever-changing conditions of ambient illumination. A major contributor to this adaptation is the light-driven translocation of key signaling proteins into and out of ROS. The present review shows how generation of the second lipid messengers from phosphatidylcholine, phosphatidic acid, and diacylglycerol is modulated by the different illumination states in the vertebrate retina. Findings suggest that the light-induced translocation of phototransduction proteins influences the enzymatic activities of phospholipase D, lipid phosphate phosphatase, diacylglyceride lipase, and diacylglyceride kinase, all of which are responsible for the generation of the second messenger molecules.

α‐Luminol prevents decreases in glutamate, glutathione, and glutamine synthetase in the retinas of glaucomatous DBA/2J mice

To test the hypothesis that in DBA/2J mice, oxidative stress decreases glutamine synthetase (GS) levels resulting in a loss of neuronal glutamate and that the antioxidant alpha-luminol (GVT) decreases this stress and glutamate loss in some types of glaucoma. DBA/2J mice were separated into two groups, of which one was not treated, and the other treated with GVT in the drinking water. At 7 months of age, retinas were examined from five untreated DBA/2J mice, seven GVT-treated mice, and five C57BL/6 mice (negative controls). Serial 0.5 microm plastic sections were immunogold stained for glutamate, GS, and total glutathione, followed by image analysis for staining patterns and density. Focal decreases in glutamate immunostaining were common in the inner nuclear layer (INL) of DBA/2J retinas, but not in C57BL/6 or GVT-treated DBA/2J retinas. Decreases in glutathione and GS immunostaining were found in DBA/2J retinal regions where neuronal glutamate immunostaining was reduced. Retinas from GVT-treated DBA/2J had no significant decreases in INL levels of glutamate, glutathione, or GS. Retinas of dogs with primary glaucoma are reported to have focal depletion of neuronal glutamate. In DBA/2J mice, similar changes occur prior to the development of clinical disease. In these focal glutamate-depleted regions, levels of glutathione and GS are also reduced, consistent with the hypothesis that oxidative stress contributes to retinal changes in glaucoma. The ability of GVT, an antioxidant, to inhibit retinal abnormalities in DBA/2J mice provides further support for this hypothesis.

ZBED4, a BED-Type Zinc-Finger Protein in the Cones of the Human Retina

To characterize the ZBED4 cDNA identified by subtractive hybridization and microarray of retinal cone degeneration (cd) adult dog mRNA from mRNA of normal dog retina. The cDNA library obtained from subtractive hybridization was arrayed and screened with labeled amplicons from normal and cd dog retinas. Northern blot analysis was used to verify ZBED4 mRNA expression in human retina. Flow cytometry sorted peanut agglutinin (PNA)-labeled cones from dissociated mouse retinas, and quantitative RT-PCR (QPCR) was used to measure ZBED4 mRNA levels in these cone cells. Immunohistochemistry localized ZBED4 in human retinas. Expression of ZBED4 mRNA transiently transfected into HEK293 cells was analyzed by immunofluorescence. ZBED4 subcellular localization was determined with Western blot analysis. One of 80 cDNAs differentially expressed in normal and cd dog retinas corresponded to a novel gene, ZBED4, which is also expressed in human and mouse retinas. ZBED4 mRNA was found to be present in cone photoreceptors. When ZBED4 cDNA was transfected into HEK293 cells, the expressed protein showed nuclear localization. However, in human retinas, ZBED4 was localized to cone nuclei, inner segments, and pedicles, as well as to Müller cell endfeet. Confirming these immunohistochemical results, the 135-kDa ZBED4 was found in both the nuclear and cytosolic extracts of human retinas. ZBED4 has four predicted DNA-binding domains, a dimerization domain, and two LXXLL motifs characteristic of coactivators/corepressors of nuclear hormone receptors. ZBED4 cellular/subcellular localization and domains suggest a regulatory role for this protein, which may exert its effects in cones and Müller cells through multiple ways of action.

Detection of Intact rAAV Particles up to 6 Years After Successful Gene Transfer in the Retina of Dogs and Primates

Gene transfer to the retina using recombinant adeno-associated viral (rAAV) vectors has proven to be an effective option for the treatment of retinal degenerative diseases in several animal models and has recently advanced into clinical trials in humans. To date, intracellular trafficking of AAV vectors and subsequent capsid degradation has been studied only in vitro, but the fate of AAV particles in transduced cells following subretinal injection has yet to be elucidated. Using electron microscopy and western blot, we analyzed retinas of one primate and four dogs that had been subretinally injected with AAV2/4, -2/5, or -2/2 serotypes and that displayed efficient gene transfer over several years. We show that intact AAV particles are still present in retinal cells, for up to 6 years after successful gene transfer in these large animals. The persistence of intact vector particles in the target organ, several years postadministration, is totally unexpected and, therefore, represents a new and unanticipated safety issue to consider at a time when gene therapy clinical trials raise new immunological concerns.

Steroids Do Not Prevent Photoreceptor Degeneration in the Light-Exposed T4R Rhodopsin Mutant Dog Retina Irrespective of AP-1 Inhibition

AP-1 has been proposed as a key intermediate linking exposure to light and photoreceptor cell death in rodent light-damage models. Inhibition of AP-1 associated with steroid administration also prevents light damage. In this study the role of steroids in inhibiting AP-1 activation and/or in preventing photoreceptor degeneration was examined in the rhodopsin mutant dog model. The dogs were dark adapted overnight, eyes dilated with mydriatics; the right eye was light occluded and the fundus of the left eye photographed ( approximately 15-17 overlapping frames) with a fundus camera. For biochemical studies, the dogs remained in the dark for 1 to 3 hours after exposure. Twenty-four hours before exposure to light, some dogs were treated with systemic dexamethasone or intravitreal/subconjunctival triamcinolone. AP-1 DNA-binding activity was determined by electrophoresis mobility shift assay (EMSA) and phosphorylation of c-Fos and activation of ERK1/2 were determined by immunoblot analyses. The eyes were collected 1 hour and 2 weeks after exposure to light, for histopathology and immunocytochemistry. Inhibition of AP-1 activation, and phosphorylation of ERK1/2 and c-Fos were found after dexamethasone treatment in light-exposed T4R RHO mutant dog retinas. In contrast, increased AP-1 activity and phosphorylation of c-Fos and ERK1/2 were found in triamcinolone-treated mutant retinas. Similar extensive rod degeneration was found after exposure to light with or without treatment, and areas with surviving photoreceptor nuclei consisted primarily of cones. Only with systemic dexamethasone did the RPE cell layer remain. Intraocular or systemic steroids fail to prevent light-induced photoreceptor degeneration in the T4R RHO dog retina. Finding that systemic dexamethasone prevents AP-1 activation, yet does not prevent retinal light damage, further supports the hypothesis that AP-1 is not the critical player in the cell-death signal that occurs in rods.

25. Detection of Intact rAAV Particles up to 6 years after Successful Gene Transfer in the Retina of Dogs and Primates

25. Detection of Intact rAAV Particles up to 6 years after Successful Gene Transfer in the Retina of Dogs and Primates

Operating in the Dark

A standard operating microscope was modified with a bandpass infrared filter in the light path and infrared image intensifiers for each of the 2 eyepieces. We evaluated this system for subretinal injections in normal control dogs and those with a mutation in the rhodopsin gene. Rhodopsin-mutant dogs are a model for human autosomal dominant retinitis pigmentosa, and their retinas degenerate faster when exposed to modest light levels as used in routine clinical examinations. We showed that the mutant retinas developed severe generalized degeneration when exposed to the standard operating microscope light but not the infrared light. The modified operating microscope provided an excellent view of the ocular fundus under infrared illumination and allowed us to perform subretinal injections in the retinas of the rhodopsin-mutant dogs without any subsequent light-induced retinal degeneration.

Targeting gene expression to cones with human cone opsin promoters in recombinant AAV

Specific cone-directed therapy is of high priority in the treatment of human hereditary retinal diseases. However, not much information exists about the specific targeting of photoreceptor subclasses. Three versions of the human red cone opsin promoter (PR0.5, 3LCR-PR0.5 and PR2.1), and the human blue cone opsin promoter HB569, were evaluated for their specificity and robustness in targeting green fluorescent protein (GFP) gene expression to subclasses of cones in the canine retina when used in recombinant adeno-associated viral vectors of serotype 5. The vectors were administered by subretinal injection. The promoter PR2.1 led to most effective and specific expression of GFP in the long- and medium-wavelength-absorbing cones (L/M cones) of normal and diseased retinas. The PR0.5 promoter was not effective. Adding three copies of the 35-bp LCR in front of PR0.5 lead to weak GFP expression in L/M cones. The HB569 promoter was not specific, and GFP was expressed in a few L/M cones, some rods and the retinal pigment epithelium. These results suggest that L/M cones, the predominant class of cone photoreceptors in the retinas of dogs and most mammalian species can be successfully targeted using the human red cone opsin promoter.

Clinical Light Exposure, Photoreceptor Degeneration, and AP-1 Activation: A Cell Death or Cell Survival Signal in the Rhodopsin Mutant Retina?

The T4R RHO mutant dog retina shows retinal degeneration with exposures to light comparable to those used in clinical eye examinations of patients. To define the molecular mechanisms of the degeneration, AP-1 DNA-binding activity, composition, posttranslational modification of the protein complex, and modulation of ERK/MAPK signaling pathways were examined in light-exposed mutant retinas. Dark-adapted retinas were exposed to short-duration light flashes from a retinal camera used clinically for retinal photography and were collected at different time points after exposure. Electrophoretic mobility shift assay (EMSA), supershift EMSA, Western blot analysis, and immunocytochemistry were used to examine AP-1 signaling. Exposure to light of mutant retinas significantly increased AP-1 DNA-binding activity by 1 hour after exposure, and levels remained elevated for 6 hours. Shielded mutant retinas had similar AP-1 levels to shielded or exposed wild-type retinas. The parallel phosphorylation of c-Fos and activation of ERK1/2 was detected only in exposed mutant retinas. Exposure to light changed the composition of the AP-1 protein complex in the mutant retina from c-Jun/Fra-1/c-Fos to JunB/c-Fos. Immunohistochemistry showed that the components of activated AP-1 (JunB, and phosphorylated c-Fos, and phosphorylated ERK1/2 isoforms) were localized in Müller cells. The inner nuclear layer/Müller cell localization of the key proteins induced by light exposure raises the question of the direct involvement of AP-1 in mediating photoreceptor cell death in this model of autosomal dominant retinitis pigmentosa.

Microvessel loss, vascular damage and glutamate redistribution in the retinas of dogs with primary glaucoma

Vascular damage and ischemia-like changes in glutamate distribution occur in primary glaucoma (PG) in dogs. We measured the microvessel density in PG retinas to determine whether microvessel loss may induce ischemia and glutamate redistribution. Sections from 12 control and 33 glaucomatous dog retinas. Vessels in retinas were identified by staining with Griffonia simplicifolia isolectin B4 or immunohistochemical staining for laminin or glutamate. Damage to regions of the inner nuclear layer (INL) was classified as mild (< 10% damaged neurons), moderate (> or = 10% damaged neurons, INL > or = 2 cells thick) or severe (INL < 2 cells thick). Glutamate redistribution was found in some mildly damaged regions and increased as damage increased. Regions with increased glutamate redistribution and increased damage had lower densities of microvessels in plastic sections. However, neuronal damage and glutamate redistribution were seen even in areas adjacent to the remaining microvessels. Microvessel loss in damaged regions was confirmed in paraffin sections with lectin staining and immunohistochemical localization of laminin. The density of larger vessels was not decreased in PG, but larger vessels often had thickened walls, cuffing with leukocytes, and leakage of albumin. Microvessel loss may occur in regions of glutamate redistribution and neuronal damage in PG retinas. Larger vessels were often damaged. The redistribution of glutamate is associated with a loss of microvessels, even in mildly damaged regions. However, neuronal damage and glutamate redistribution may occur close to remaining microvessels, suggesting microvessel loss was not the sole factor inducing these changes.

Retrograde axonal transport obstruction of brain‐derived neurotrophic factor (BDNF) and its TrkB receptor in the retina and optic nerve of American Cocker Spaniel dogs with spontaneous glaucoma

To determine the degree of retrograde optic nerve axonal transport obstruction at the scleral lamina cribosa level by examining levels of brain-derived neurotrophic factor (BDNF) and its tyrosine kinase receptor type B (TrkB) in dogs presenting with high intraocular pressure. A total of 10 eyes, four normal and six glaucomatous eyes, from normal and affected American Cocker Spaniels with primary glaucoma were studied. All eyes were assessed by neuro-ophthalmic examination, tonometry, gonioscopy, slit-lamp biomicroscopy, and indirect ophthalmoscopy prior to enucleation. Immunocytochemistry analysis was performed to evaluate BDNF and TrkB receptor expression in retina and optic nerve in normal and glaucomatous dogs. In all normal eyes BDNF immunostaining was detected in the cytoplasm of retinal ganglion cells (RGC), inner plexiform layer (IPL), inner nuclear layer (INL), nerve fiber layer (NFL), optic nerve head cells, and lamina cribosa cells. In all glaucomatous eyes BDNF was more evident in RGC, NFL and lamina cribosa cells. TrkB receptor was detected in the cytoplasm of RGC, NFL and ONH bundles in all normal eyes, and in a more intense pattern in all glaucomatous eyes. BDNF retrograde axonal transport is substantially inhibited by intraocular pressure elevation. TrkB accumulation at the ONH in glaucoma suggests a role for neurotrophin deprivation in the pathogenesis of RGC death in canine glaucoma, as well as a possible paracrine and/or autocrine signaling within the lamina cribosa. Neurotrophin signaling may regulate more than neuronal development, survival and differentiation. BDNF neurotrophin and its TrkB receptor expression by lamina cribosa cells and ONH astrocytes in glaucomatous eyes may help to determine the role of these cells as a paracrine source in terms of retinal ganglion cell survival, during episodes of elevated intraocular pressure.

The in vitro protective effect of α-tocopherol on oxidative injury in the dog retina

Oxidative stress is a risk factor for eye diseases. Free radicals elicited during the inflammatory process often lead to oxidative damage of lipids (lipid peroxidation). The retina is highly vulnerable because of its high content of polyunsaturated fatty acids (PUFAs). The aim of this study was to investigate in vitro the effect of α-tocopherol on the Fe 2+-ascorbate induced lipid peroxidation in the canine retina. Lipid peroxidation of retinal homogenates was carried out with and without the addition of α-tocopherol and monitored both by chemiluminescence and production of thiobarbituric acid reactive substances (TBARS). Total chemiluminescence counts per minute was lower in those homogenates pre-incubated with α-tocopherol. Thus, with 1 μmol α-tocopherol/mg of protein, 100% inhibition of chemiluminescence and a decrease of TBARS content from 20.46 ± 0.85 to 2.62 ± 2.77 nmol/mg protein were observed. Simultaneously, changes produced by oxidative stress were noted in the fatty acid composition of retinal lipids. Docosahexaenoic acid was decreased from 14.33 ± 2.32% to 1.84 ± 0.14% after peroxidation, but this fatty acid remained unaltered in the presence of 1 μmol α-tocopherol. These results show that under these experimental conditions, α-tocopherol may act as anti-oxidant protecting retinal membranes from deleterious effects. Further studies are required to assess its use in free radical generating conditions affecting the canine retina.

Canine and Human Visual Cortex Intact and Responsive Despite Early Retinal Blindness from RPE65 Mutation

BackgroundRPE65 is an essential molecule in the retinoid-visual cycle, and RPE65 gene mutations cause the congenital human blindness known as Leber congenital amaurosis (LCA). Somatic gene therapy delivered to the retina of blind dogs with an RPE65 mutation dramatically restores retinal physiology and has sparked international interest in human treatment trials for this incurable disease. An unanswered question is how the visual cortex responds after prolonged sensory deprivation from retinal dysfunction. We therefore studied the cortex of RPE65-mutant dogs before and after retinal gene therapy. Then, we inquired whether there is visual pathway integrity and responsivity in adult humans with LCA due to RPE65 mutations (RPE65-LCA).Methods and Findings RPE65-mutant dogs were studied with fMRI. Prior to therapy, retinal and subcortical responses to light were markedly diminished, and there were minimal cortical responses within the primary visual areas of the lateral gyrus (activation amplitude mean ± standard deviation [SD] = 0.07% ± 0.06% and volume = 1.3 ± 0.6 cm3). Following therapy, retinal and subcortical response restoration was accompanied by increased amplitude (0.18% ± 0.06%) and volume (8.2 ± 0.8 cm3) of activation within the lateral gyrus (p < 0.005 for both). Cortical recovery occurred rapidly (within a month of treatment) and was persistent (as long as 2.5 y after treatment). Recovery was present even when treatment was provided as late as 1–4 y of age. Human RPE65-LCA patients (ages 18–23 y) were studied with structural magnetic resonance imaging. Optic nerve diameter (3.2 ± 0.5 mm) was within the normal range (3.2 ± 0.3 mm), and occipital cortical white matter density as judged by voxel-based morphometry was slightly but significantly altered (1.3 SD below control average, p = 0.005). Functional magnetic resonance imaging in human RPE65-LCA patients revealed cortical responses with a markedly diminished activation volume (8.8 ± 1.2 cm3) compared to controls (29.7 ± 8.3 cm3, p < 0.001) when stimulated with lower intensity light. Unexpectedly, cortical response volume (41.2 ± 11.1 cm3) was comparable to normal (48.8 ± 3.1 cm3, p = 0.2) with higher intensity light stimulation.ConclusionsVisual cortical responses dramatically improve after retinal gene therapy in the canine model of RPE65-LCA. Human RPE65-LCA patients have preserved visual pathway anatomy and detectable cortical activation despite limited visual experience. Taken together, the results support the potential for human visual benefit from retinal therapies currently being aimed at restoring vision to the congenitally blind with genetic retinal disease.

Age-Dependent Retinal Capillary Pericyte Degeneration in Galactose-Fed Dogs

The galactose-fed beagle develops diabetes-like microvascular changes that are histologically and clinically similar in appearance to all stages of human diabetic retinopathy. This animal model is extremely useful for evaluating drugs for the treatment of diabetic retinopathy; however, the time required to develop the various retinal lesions (24-72 months for background to the proliferative stage) may be considered prohibitive. Retinal vascular changes begin with an initial degeneration of capillary pericytes, which has been linked to the aldose reductase catalyzed formation of galactitol. Because aldose reductase-linked sugar cataract formation is known to be age dependent, with the onset and severity of cataract higher in younger diabetic and galactose-fed animals, retinal capillary changes in the eyes of initially 2- versus 9-month-old beagles fed a diet containing 30% galactose were compared. Eyes were enucleated after 36 months of galactose feeding, the intact retinal capillaries were isolated by trypsin digestion, and defined retinal regions were evaluated by computer image analysis. Nicotinamide adenine dinucleotide phosphate-dependent reductase activity, using DL-glyceraldehyde and D-xylose as substrates, was also compared in the lenses and whole retinas of eyes from the 2- and 9-month-old beagles. Significantly (P<or=0.05) increased pericyte degeneration, expressed as either the number of pericytes/mm capillary length or the ratio of endothelial cells versus pericytes (E/P ratio) was observed in the retinas of the younger dogs. The number of microaneurysms per eye was also significantly increased in the younger dogs, but no difference in acellular capillary areas was observed. This correlates with a threefold higher level of reductase activity in the retinas of the 2-month-old dogs. Because retinal capillary pericyte destruction is age dependent similar to the formation of sugar cataracts, the use of younger dogs may shorten the time period required for evaluating the efficacy of drugs for diabetic retinopathy in this animal model.

Genetic heterogeneity of day blindness in Alaskan Malamutes

Day blindness is a progressive and specific degeneration of cone photoreceptors in the retina of young dogs. This disorder has been associated with a breed-specific non-synonymous substitution in exon 6 of the cyclic nucleotide gated channel beta3 (CNGB3) gene in German Shorthaired Pointer dogs and a genomic deletion removing the entire gene in Alaskan Malamute dogs from the USA. To further investigate this disorder, we characterized CNGB3 in a three-generation pedigree of Alaskan Malamute dogs from Australia segregating for day blindness. Fifteen of the dogs showed clinical signs of day blindness. Four of these were definitively diagnosed by standardized electroretinography. Polymerase chain reaction amplification of exon 6 of CNGB3 was attempted, and as expected, amplification was successful in the 18 unaffected or carrier dogs. However, a non-mutated exon 6 was also amplified and sequenced in six of the 15 affected dogs. On sequencing each exon and exon/intron boundary in two such affected individuals and two unaffected individuals, three exonic substitutions and 12 intronic changes were noted. These sequence variations in affected individuals were also present in one or both unaffected dogs and so appear to have no obvious effect on the protein's function. Hence, day blindness shows genetic heterogeneity within the Alaskan Malamute population of Australia, a result that is somewhat unexpected given the relatively small effective population size of this breed.

Canine retinal angioblasts are multipotent

The purpose of this study was to culture and characterize endothelial cells and angioblasts, vascular precursors, from adult and neonatal dog retina and determine if angioblasts are committed to endothelial cell lineage or have the potential to be multipotent, i.e. express phenotypic characteristics of other vascular cell types. Endothelial cells were established from adult dog retina (ADREC) by the technique of Gitlin and D'Amore. For angioblasts, pieces of neonatal day 2 (P2) avascular peripheral retina were placed under coverslips until sufficient cells had explanted. All cells were maintained initially on hyaluronic acid (HA)/fibronectin (FN) substratum. Neonatal canine retinal angioblasts (NCRA) were maintained initially on retinal-derived growth factor with alpha-amino adipic acid to inhibit growth of Muller cells. Cell lines were characterized by enzyme histochemistry [menadione-dependent alpha glycerophosphate dehydrogenase (αGPDH), marker for angioblasts] and immunocytochemistry. Once characterized, cells were grown on FN, or collagens I or IV substrata and fed platelet-derived growth factor-BB (PDGF-BB) or fibroblast growth factor-2 (FGF-2). The phenotypic expression of a marker for endothelial cells [acetylated LDL (acLDL) uptake] or a marker for pericytes and smooth muscle cells, production of alpha smooth muscle actin (αSMA), was evaluated under those conditions. The canine retinal cell lines that were established had the following characteristics when maintained on serum and a retinal extract. Angioblasts had low expression of vWf and VEGF-R2 (two markers for canine endothelial cells), and very low uptake of acLDL but high expression of αGPDH and adenosine A2a receptors (A2aR) (two markers for canine angioblasts in vivo). ADREC had high expression of endothelial cell markers (vWf, VEGF-R2, and acLDL uptake) but minimal expression of αGPDH and A2aR. Both angioblasts and endothelial cells expressed CXCR4, a marker for hemangioblasts. Angioblasts grown on any of the substrata in the presence of FGF-2 had high uptake of acLDL and low expression of αSMA, while those grown in the presence of PDGF-BB had high expression of αSMA and low uptake of acLDL. In conclusion, angioblasts cultured from peripheral vascular retina have low expression of endothelial cell markers and high αGPDH and A2aR, markers for canine angioblasts in vivo. Angioblasts will internalize acLDL when maintained on FGF-2 and express αSMA when maintained on PDGF-BB, suggesting that they have the potential to become endothelial cells or pericytes, i.e. are multipotent.

881. Safety, Efficacy and Biodistribution of Recombinant AAV2-RPE65 Vector Delivered by Ocular Subretinal Injection

Top of pageAbstract Introduction: AAV2 delivery of the RPE65 cDNA to the retina of blind RPE65-deficient dogs or rd12 mice restores vision as determined electrophysiologically and behaviorally. This strategy is being considered for human trials in RPE65-associated Leber congenital amaurosis, but safety and dose-efficacy within a non-toxic range with this vector have not been defined. Here we report the results of toxicology and biodistribution studies in two mammalian species, dogs and rats. Methods: Vector related pathology and spread after subretinal delivery of AAV2-CBA-RPE65 was studied in RPE65-mutant dogs and normal Spague-Dawley rats. Doses of vector delivered bracketed those intended for the clinical study. Results: There was no systemic toxicity at any vector dose in either species. In dogs ocular examinations showed mild or moderate inflammation that resolved over an initial 3-month period. Retinal histopathology indicated that traumatic lesions from the injection were common, but retinal thinning within the injection region only occurred at the highest vector doses. Biodistribution studies were also performed in RPE65-mutant dogs at various times after vector injection and in normal rats at about 2 weeks and 2 months post-injection. Vector DNA was not widespread outside the injected eye with blood and gonadal tissue consistently negative. Concomitant dose-response results in the RPE65-mutant dogs indicated that the highest 1.5 log unit range of vector doses was efficacious. Conclusions: Given the efficacy and toxicity limits defined in this study, a range for safe vector dose escalation of subretinal AAV2-CBA-RPE65 is suggested for initial human trials.